Replicons of pestviruses that do not express c and or e1 protein and infectious viral patricles containing same, that can be used in vaccines

ABSTRACT

The present invention provides new Pestiviral RNA genomes (replicons) that are able to replicate, and can be packaged into infectious viral particles in cells that complement the missing protein(s), but do not produce infectious progeny virus. Such replicons can be useful for vaccine purposes. The replicons encode most, preferably all, envelop proteins of the virus, while, on the other hand, it would not be capable of producing infectious progeny virus. The present invention provides a Pestiviral replicon, preferably from the Bovine Viral Diarrhea Virus (BVDV), which expresses all structural proteins except for a functional C or E1 protein. Preferably at least part of the coding sequence of the E1 or C protein has been deleted from said replicon. The present inventors proved for the first time, that both C and E1 structural proteins are essential for the formation of infectious pestiviruses. Furthermore it was shown that deletion of C and E1 does not impact the ability of RNA self-replication. By using cell lines constitutively expressing pestiviral structural proteins, Capsid- or E1-proteins can be efficiently trans-complemented. The resulting virions are able to infect bovine target cells and to transfer the replicons without generating replication-competent virus progeny. In other words, no infectious progeny virus is produced. The complemented virions are indistinguishable from wild-type Pestivirus in virus neutralization experiments. Recombinations yielding infectious wild-type virus were not detected in any of the complementation experiments. The complemented viruses may be used for the safe and efficacious immunization against BVDV.

The present invention relates to replicons of Pestiviruses that do notexpress all structural proteins of the virus, infectious viral particlescontaining said replicons, a method for producing said infectious viralparticles and vaccines containing said viral particles.

Animals may be protected against pestiviruses by vaccination, however,conventional inactivated or modified live vaccines have disadvantagesconcerning safety as well as efficacy. Therefore, new types of vaccinesshould be developed.

Pestiviruses can be divided into two different biotypes, cytopathogenic(cp) and non cytopathogenic (ncp) viruses, respectively. Bovine viraldiarrhea virus (BVDV), a member of the genus Pestivirus within thefamily Flaviviridae is the causative agent of bovine viral diarrhea, aneconomically important disease of cattle world-wide. Genetically andstructurally closely related virus species are Classical Swine FeverVirus (CSFV) and the ovine Border Disease Virus (BDV). Pestiviruses caninduce severe diseases with marked economical losses world wide. Themajor economic losses caused by BVDV infections are reduced fertility,abortions and the generation of persistently infected calves, which candevelop fatal “Mucosal Disease”.

While cp BVDV induce apoptosis and cell death and express non-structuralprotein 3 (NS3), inoculation with ncp BVDV leads to persistent infectionof cell cultures and NS3-expression is not detectable. The pestivirusgenome consists of a single-stranded RNA of positive orientation. TheRNA has a length of approximately 12.3 kb and contains one large openreading frame (ORF), which is flanked by non-translated regions (NTR) atboth genome ends. The pestiviral ORF is translated into one polyprotein,which is co- and post-translationally processed into 11 (ncp BVDV) or 12(cp BVDV) mature proteins by viral and cellular proteases. Pestivirusvirions consist of four structural proteins, a capsid (C) protein andthree glycosylated envelope proteins (E^(RNS), E1, E2). BVDV antibodiesare directed against E^(RNS), E2 and NS3. Neutralizing activity waspredominantly demonstrated for E2-specific antibodies.

Studies on the replication of pestiviruses have been considerablyfacilitated by reverse genetic systems and the discovery of autonomouslyreplicating subgenomic RNAs (replicons) (4, 26).

Pestivirus genomes with deletions were first described as defectiveinterfering particles (DI) for BVDV and CSFV (24, 27).

Since then many reports relating to the replication of pestiviruses havebeen published. Trans-complementation of structural proteins of virusesof the family Flaviviridae has been reported.

Pestivirus self-replicating RNAs are important tools for anunderstanding of virus replication, assembly and egress.Trans-complementation of deleted parts of the genome can be used, forexample, for the identification of trans-acting elements of a pestiviralgenome.

The minimal requirements for CSFV replication were investigated, forexample, by creating defective CSFV genomes lacking the gene sequencesfor the structural proteins. It was found that the defective CSFVgenomes still replicated and could be packaged into viral particles whenintroduced in SK-6 cells together with helper A187-CAT RNA (Moser etal., J. Virol., 7787-7794, 1999). An autonomously replicating defectiveBVDV genome which lacks the genes encoding C, E^(ms), E1, E2, p7 and NS2had been described (Behrens et al., J. Virol., 72, 2364-2372, 1998). Forthe Kunjin virus (KUN) replicons with deletions in the structuralregions were packaged into virons using a system where BHK-21 cells weretransfected by two consecutive electropoartions, first with the mutatedKUN replicons, and subsequently with a recombinant Semlike Forest Virusreplicon expressing KUN structural proteins (Khromykh et al., J. Virol.,72(7), 5967-5977, 1998). For CSFV it had been recognized thattrans-complemented defective virions that contained E^(ms) deletedreplicons could potentially serve as a basis for vaccines. CSFV,E^(RNS)-deleted replicons were generated and trans-complemented using aswine kidney cell line (SK6) constitutively expressing CSFV E^(RNS). Theresulting virions were able to infect SK6 cells without the productionof infectious virus progeny and could be passaged on E^(RNS)-expressingSK6 cells. Pigs were protected against lethal CSFV challenge afterimmunization with the complemented virions (40).

So far, there are only few reports concerning trans-complementation ofBVDV replicons. Defects in the coding regions for non-structuralproteins NS3, NSNS4a, NS4B and NS5B were found to be non complementable,whereas defects in the NS5A unit could be complemented in trans(Grassmann et al., J. Virol., 75, 7791-7802, 2001). (4, 18, 36)Trans-complementation of BVDV E2 and/or p7, using cell linesconstitutively expressing BVDV E2 and/or p7 for complementation havealso been described (Harada et al., J. Virol., 74, 9498-9506, 2000).

The present invention provides new Pestiviral RNA genomes (replicons)that are able to replicate, and can be packaged into infectious viralparticles in cells that complement the missing protein(s), but do notproduce infectious progeny virus. Such replicons can be useful forvaccine purposes.

Although it was known in the art that all the genes encoding structuralproteins of a Pestivurs can be deleted without affecting the ability toreplicate (Behrens et al, supra), such a mutant, missing all structuralproteins would not be suitable for vaccine purposes. A vaccine mainlyaims at invoking an immune response. A vaccine, on the one hand, shouldbe able to elicit a protective immune response, while, on the otherhand, it should of course not invoke the (viral) disease in theinoculated animal or contact animals. The immune response induced isusually mainly directed against the envelop proteins of the virus. But,if a replicon would be used from which all the structural, moreparticular, all the envelop protein coding sequences have been deleted,such proteins would not be produced from the replicon and no immuneresponse to these proteins would be obtained.

The present inventors aimed at providing a replicon that would stillencode most, preferably all, envelop proteins of the virus, while, onthe other hand, it would not be capable of producing infectious progenyvirus.

The present invention provides a replicon of a Pestivirus, preferablythe Bovine Viral Diarrhea Virus (BVDV), which expresses all structuralproteins of BVDV except for a functional C and/or E1 protein. Preferablyat least part of the coding sequence of the E1 or C protein has beendeleted from said replicon.

A replicon is a self-replicating RNA molecule. In this case the RNAmolecule is the RNA genome of the BVD virus. Replicons can beconstructed from a full length c-DNA clone of the BVD virus, for examplefrom a full-length BVDV cDNA clone like pA/BVD/ins- (Meyers et al., J.Virol. 70:8606-8613, 1996). The BVDV RNA contains one ORF that istranslated into one polyprotein, which is processed into mature proteinsby viral and cellular proteases. In constructing mutated replicons careshould be taken not to introduce a frameshift within the BVDV ORF.Furthermore, when coding sequences for structural proteins are deleted,the sequences encoding some N and/or C terminal amino acids may beretained to ensure proper post translational processing of the precursorpolypeptide (protease cleavage sites are retained), For example, thecapsid (C protein) of BVDV strain NCP7 is encoded by amino acids (AA)169-270. About 25 amino acids at the C terminus (M245270) function as asignal sequence for the translocation of the E^(RNS)E1E2 polyprotein,and as a putative anchor of the capsid protein. The signal sequence atthe C terminus of the capsid protein is essential for a furtherprocessing of the polyprotein downstream.

In case where expression of a functional C protein is prevented bydeleting part of the gene encoding the C protein, the coding sequenceencoding the part of the protein essential for further downstreamprocessing should preferably be retained. For the C protein a about 32AA should preferably be retained at the N terminus. The exact length ofthe sequences necessary for downstream processing may vary per strain orcell type.

The envelop protein E1 of BVDV strain NCP7 is encoded by amino acids 498to 692. About 33 amino acids at the C terminus (AA 660 to 692) functionas a signal sequence for the translocation of the E2 envelop protein,and as a putative anchor of the E1 envelope protein.

However, other signal sequences or the analogous sequences of otherpestivirus species (e.g. CSFV) may be used instead of, for example, theBVDV sequence. For E1 mutants of BVDV no M at the N terminus have anyfunction as a signal sequence.

For both E1 and C mutants several ways for construction of replicons areconceivable, that do not express the functional proteins. For example,such mutations may be created by partial deletions whereby the signalsequences are retained, or partial or complete deletions whereby thesignal sequences are replaced by analogous signal sequences, ormutations where only base pair exchanges are made.

The present inventors proved for the first time, that both C and E1structural proteins are essential for the formation of infectiouspestiviruses. Furthermore it was shown that deletion of C and E1 doesnot impact the ability of RNA self-replication.

By using cell lines constitutively expressing Pestiviral structuralproteins, Capsid- or E1-proteins can be efficiently trans-complemented.The resulting virions are able to infect target cells and to transferthe replicons without generating infectious replication-competent virusprogeny. The complemented virions are indistinguishable from wild-typePestivirus in virus neutralization experiments and can be completelyneutralized by, for example in the case of BVDV, a BVDV-specific serumas well as by a BVDV E2-specific serum. Recombinations yieldinginfectious wild-type BVDV were not detected in any of thecomplementation experiments.

The complemented viruses may be used for the safe and efficaciousimmunization against a Pestivirus, such as BVDV.

Infection of cells and animals with the trans-complemented virusesresults in replicating RNAs expressing all encoded proteins in theinfected cells but which do not generate viral particles.

Thus the complemented virions can be used for the immunization againstan infection with a pestivirus, such as BVDV, and can protect vaccinatedanimals from Pestivirus-related disease.

In a preferred embodiment a replicon according to the invention does notencode a functional C protein. This can be achieved by deleting at leastpart of the coding sequence for the C protein. In case of BVDV,preferably the coding region encoding amino acid positions 201-243 ofthe C protein is deleted.

In another embodiment of the present invention the replicon does notencode a functional E1 protein. For BVDV, preferably the coding regionencoding amino acid positions 498 to 653 of the E1 protein are deleted.

It has been found that the C protein, which is the capsid protein of thevirus, as well as the E1 protein, is essential for obtaining infectiousvirus. Mutants that lack the coding sequence for the C or the E1 proteinare thus not infectious. The advantage of mutants that do not encode acapsid or E1 protein, but do encode all (envelop) proteins responsiblefor the immune response (especially E2 and E^(ms)) is that the immuneresponse will still include a response against all envelop proteins,since the coding sequences for all envelop proteins is still present.

Likewise part of the present invention are infectious viral particles ofa pestivirus that contain a replicon according to the invention. Suchparticles can be incorporated in a vaccine for said Pestivirus, forexample BVDV.

A method for the production of viral particles according to theinvention is likewise part of the invention. Such a method may comprisethe following steps:

-   a. Providing cells that are permissive for a particular Pestivirus    (for example BVDV) and express a Pestiviral E1 and/or C protein,-   b. Transfecting said cells with a replicon according to the    invention,-   c. Culturing transfected cells obtained in step b,-   d. Harvesting the viral particles from the cell culture.

Preferably the particles produced in step d are used to infect freshcells that are permissive for a particular Pestivirus (for example BVDV)and express a Pestiviral E1 and/or C protein again.

Cells that can be used in the production of the viral particlesaccording to the invention should at least be capable of expressing theC protein and/or the E1 protein of a pestivirus, which may thepestivirus on which the replicon is based or another Pestivirus. In caseof BVDV said cells should preferably express the E1 and or C protein ofBVDV. Examples of suitable complementing cell lines are disclosed in theExamples.

In case of BVDV, such cells are preferably of bovine origin, for examplebovine kidney cells or diploid bovine esophageal cell line.

The ways by which the coding sequences of a pestivirus such as BVDV canbe introduced into said cells are known in the art. For example, thecoding sequences for the structural proteins of BVDV (at least C and/orE1) may be introduced into said cells by way of an expression plasmidencoding said proteins or by way of a helper virus. When the viralparticles are intended to be used in a vaccine the use of expressionplasmids is preferred, to avoid complicated purification methods due tothe presence of a helper virus. Suitable expression plasmids are knownin the art. After transfection of replicon RNA infectious viralparticles are produced. The cells can be grown according to methodsknown in the art until the desired viral titer is obtained. Virus may beharvested from the supernatant of the cell culture or from total celllysates.

Methods used to transfect the cells and the various cloning proceduresare known in the art (for example: Sambrock, J & Russel, D. W. MolecularCloning: A laboratory Manual (Cold Spring Harbor Press, New York, 2001).All permanent cell lines which are suitable for the replication ofpestiviruses may be used, preferentially cells of bovine origin.Preferably cells are used that can be easily transfected with DNA. Thecoding sequences of the structural proteins of most of the pestivirusescontain cryptic splicing sites. Therefore, for most of the BVDV strains,a synthetic open reading frame (synORF) eliminating the splicing sitesmay be constructed to ensure expression with promoters using the nuclearpathway (e.g. HCMV promoter). However other expression systems (viruses,replicons from other viruses, pestivirus replicons with differentdeletions) may provide the deleted proteins for trans complementation.

A vaccine containing infectious viral particles according to theinvention together with a pharmaceutically acceptable carrier islikewise part of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1.

Schematic representation of the generated constructs. Filled boxesrepresent the BVDV structural region. Dotted lines show the deletedregions and numbers indicate the nucleotide position in the BVDVfull-length RNA. Arrows indicate restriction enzyme sites flanking thedeletions. Lines at the left and right ends indicate untranslatedregions. N^(pro), autoprotease; C, capsid protein; E^(RNS), E1 and E2,envelope proteins; p7, nonstructural protein; N2 to NS5, nonstructuralproteins, 3′-UTR and 5′-UTR, noncoding regions. The scale in kb isgiven.

FIG. 2:

Detection of BVDV-specific antibodies after immunization withNCP7_ΔC_trans and challenge infection with BVDV type 1 using an indirectELISA system (A), and an NS3-blocking-ELISA (B). Mean values of theanimal groups are shown. Neutralizing titers at day 62 p. immunizationare 1:256 or higher.

FIG. 3:

Detection of BVDV-specific neutralizing antibody titers afterimmunization with NCP7_ΔC_trans. Mean values of the animal groups areshown.

FIG. 4:

Relative leukocyte counts and body temperatures after challengeinfection (mean values)

FIG. 5:

Detection of BVDV ERNS antigen with a commercial ELISA system (CheckitBVD Virus III, Dr. Bommeli AG). Mean values of the animal groups areshown.

EXAMPLES Example 1 Construction and Characterization of BVDV Replicons

After establishment of an infectious clone for BVDV CP7, a variant ofthe construct with a deletion of a 27 nucleotide insertion was generatedthat allowed recovery of ncp BVDV (26). Based on this construct termedpA/BVDV/Ins-, replicons were constructed by deletion of sequencesencoding C and E1 (FIG. 1) without introducing a frameshift within theBVDV ORF. Mutant BVDV clones shown in FIG. 1 were constructed on thebasis of the full-length cDNA clone pA/BVDV/ins-. Location ofrestriction sites are indicated by superscript numbers corresponding totheir cleavage positions in the BVDV/CP7 genome (FIG. 1).

Plasmid Constructs

The NCP7ΔC and NCP7ΔE1 plasmids were constructed by a two step cloningprocedure. Two PCR fragments were inserted into plasmid pA/BVDV/ins-digested with XhoI²⁰⁹ and KpnI^(2447,3797) (Tab. 1).

Plasmids were amplified in E. coli TOP10F′ cells (Invitrogen).Restriction enzyme digestion and cloning procedures were performedaccording to standard protocols. Plasmid DNA was purified by QIAGENPlasmid Mini, Midi or Maxi kits.

The primers used for PCR or sequencing (labeled with IRD 800) werecustom synthesized (MWG-Biotech). Primer pairs for generation of therespective constructs are summarized in Table 1.

The resulting replicons contained a deletion of a part of the Capsidregion (NCP7ΔC, amino acids position 201 to 243 (FIG. 1) or of the E1encoding region (NCP7ΔE1, amino acid position 498 to 653 (FIG. 1).

All deletions were confirmed by PCR and nucleotide sequencing of theresulting cDNA clones.

Polymerase Chain Reaction (PCR) and Sequencing

For PCR, a PTC-200 thermal cycler (MJ Research, Inc.) was used. DNAbased amplification was done by Expand High Fidelity PCR System (RocheMolecular Biochemicals) according to the supplier's protocol. ForRT-PCR, total RNA of virus-infected cells was extracted using the TRIZOLreagent (Gibco-Life Technologies).

cDNA was amplified from the RNA template by using reverse transcriptase(Promega) and a sequence-specific primer. The synthesized cDNA wasamplified with a thermostable Taq Polymerase (Promega) and PCR productswere directly sequenced. Sequencing was carried out using a ThermoSequenase Cycle Sequencing Kit (Amersham Biosciences). Nucleotidesequences were read with a LI-COR automatic sequencer (MWG Biotech) andwere analyzed using the Wisconsin software package version 9.1 (GeneticsComputer Group, USA).

In Vitro-Transcription and Electroporation

Subsequently, in vitro-transcribed full-length replicon RNA wastransfected into BVDV-negative PT cells.

In vitro-transcription of linearized full-length cDNA constructs andreplicons was performed by T7 RiboMaX™ Large Scale RNA Production System(Promega) according to the manufacturers instructions. The amount of RNAwas estimated by ethidiumbromide staining after agarose gelelectrophoresis. For transfections, 1×10⁷ PT, PT_(—)805 or KOP-R cellswere detached using a trypsine solution, washed with phosphate buffersaline without Ca⁺⁺/Mg⁺⁺(PBS⁻), mixed with 1 to 5 μg of in vitrosynthesized RNA and electroporated (two pulses at 850V, 25 μF, 156Ω)using an EasyjecT Plus (EquiBio) transfection unit.

Characterization of the Replicons

For the detection of BVDV proteins, monoclonal antibodies (mab) WB210(anti-E^(RNS), CVL, Weybridge), WB215 (anti-E2, CVL, Weybridge), CA3(anti-E2, Institute for Virology, TiHo Hannover), mab-mix WB103/105(anti-NS3, CVL, Weybridge), and C16 (anti-NS3, Institute for Virology,TiHo, Hannover) were used (15, 16, 31).

Transient expression of NS3 could be detected from 24 h posttransfection by IF for all replicons. Additionally, also E^(RNS)- andE2-specific immunofluorescence was demonstrated. However, no infectiousrecombinant BVDV could be recovered, even after serial passages andco-passages using highly susceptible KOP-R cells (Table 2). Theintensity of NS3-specific immunostaining of NCP7ΔC andNCP7ΔE1-transfected PT cells was comparable to cells transfected withfull-length NCP7 RNA and more than 85% of the cells were positive in IF(Table 2). Transfection of full-length NCP7 RNA into PT cells resultedin virus titers of up to 10³ IU/ml. As expected, electroporation ofcells with RNA of the prototype replicon DI9 (4) did not lead torecovery of infectious BVDV, but a strong immunofluorescence followingNS3 staining was observed. RT-PCR of PT cells transfected with repliconsor full-length NCP7 demonstrated presence of BVDV-specific RNA of theexpected size containing the correct deletions. In summary, deleted BVDVgenomes, which were replication-competent but lacked the ability ofgenerating virus progeny, were constructed and could efficiently betransfected into bovine cells. TABLE 1 Construction of BVDV-repliconsdeletion deletion position replicon (nt) (aa) primer sequence (5′→3′)(nt) sense ncp7ΔC  969-1095 201-243 cp7-204 AAGCCTCGAGATGCCACGTGG204-224 + (Kpnl²⁴⁴⁷ mutated) C-969R-KpnltctaggtaccTCTCTGACTCCTTAGGTGTTATC 969-947 − cp7-1096-KpngagaggtaccCTCAAGAGTCGCGCAAGAAAC 1096-1116 + p7-3804RGTCCTAGGTACCCCTGGGCA 3785-3804 − ncp7ΔE1 1860- 498-653 cp7-204AAGCCTCGAGATGCCACGTGG 204-224 + (Kpnl²⁴⁴⁷ mutated) 2333  cp7-1859R-KpnltcaggtaccGTGCATATGCCCCAAACCATGTC 1859-1838 − cp7-2334-KpnlcaacggtaccGAATTTGGACCGCTGCTACAACC 2334-2354 + p7-3804RGTCCTAGGTACCCCTGGGCA 3785-3804 −

TABLE 2 Replication and trans-complementation of BVDV NCP7 RNAs within-frame deletions (replicons). Titer of virus progeny^(c)replication^(a) 1st passage^(b) 24 h after transfection of 4thpassage^(d) after RNA transfection in (24 h, 48 h, 72 h) complementingof virus progeny construct PT cells (24 h) on KOP-R-cells PT_805 cells(IU/ml) on KOP-R cells NCP7 ++ (P)^(e) + (10³ IU/ml)   10^(7.3) (P) +(P) NCP7ΔC ++^(f) Ø^(f) >10^(5.0) Ø NCP7ΔE1 ++ Ø >10^(5.0) Ø DI9 +++ Ø<10⁰ nd^(a)PT cells were transfected with in vitro-transcribed RNA andIF-stained for NS3-expression after 24 h.^(b)KOP-R cells were inoculated with undiluted supernatants 24 h, 48 hand 72 h after transfection of PT cells (1 ml per 10⁵ cells). KOP-Rcells were stained with NS3-specific mabs 5 days post inoculation.^(c)PT_805 cells were transfected with in vitro-transcribed RNA and thesupernatants were titrated after 24 h using KOP-R-cells.^(d)KOP-R cells were inoculated using supernatants of transfected PT_805cells with a titer >10⁶ IU/ml (1 ml per 10⁵ cells).^(e)P indicates the detection of virus plaques after NS3-staining (>10cells).^(f)Ø = no BVDV-specific immunofluorescence; + = weak positiveNS3-IF-signal and <50% IF-positive cells, ++ = positiveNS3-IF-signal >50% IF-positive cells, +++ = strong positiveNS3-IF-signal and >50% IF-positive cells.^(g)nd = not done

Example 2 Establishment of C-E^(ms)-E1-E2 Expressing Cells

Establishment of C-E^(RNS)-E1-E2 expressing PT cells In order to docomplementation studies with the BVDV replicons, a bovine cell line wasestablished that constitutively expresses BVDV structural proteins.

PT cells (RIE11 CCVL), a permanent bovine kidney cell line, was obtainedfrom the collection of cell lines in veterinary medicine at the FederalResearch Center of Virus diseases of Animals, Insel Riems (CCLV). PTcells were chosen due to their applicability for DNA/RNA transfectionand for generation of constitutively expressing cell lines. Cells weregrown in Dulbecco's minimal essential medium (DMEM) supplemented with10% BVDV-free fetal bovine serum (FBS).

The genomic region encoding the structural proteins (C-E^(RNS)-E1-E2) ofBVDV strain PT810 (41) was cloned as a chemically synthesized syntheticopen reading frame (Syn-ORF; kindly provided by Tobias Schlapp, BayerAG, Leverkusen). It consisted of 2694 nucleotides extending fromnucleotide 890 to 3584 of the nucleotide sequence of BVDV strain NADL(9) and was inserted into the pcDNA3.1 expression plasmid (Invitrogen)using restriction sites KpnI and NotI. The nucleotide sequence ofSyn-ORF had been changed to remove splice sites (35), but retained theoriginal amino acid sequence of BVDV strain PT810. Additionally, thefirst codon of Syn-ORF was changed to a methionin to allow expression ofthe polyprotein under the control of the HCMV immediate early promotorpresent in pcDNA3.1, and a stop codon was inserted behind the lastcodon.

The resulting construct pcDNA_C-E2 (1 μg) was used to transfect PT cellswith the SUPERFECT reagent (Qiagen). At 2 days after transfection, cellculture medium was changed to DMEM supplemented with 10% bovine serumand 1 mg of G418 (Gibco, Life Technologies) per ml. G418-resistantcolonies were isolated, and replated several times.

After G418 selection and passaging, resistant cell clones were testedfor BVDV E^(RNS) and E2 expression using mabs WB210 and WB215,respectively. Positive cells were cloned, passaged under G418 selectionand tested again for E_(RNS) and E2 expression. In the IF analyses,G418-resistant cell clone PT_(—)805 contained more than 90% E^(RNS) andE2 expressing cells. During further passaging of PT_(—)805 cells for 6months in the presence of G418, the portion of E^(RNS) and E2 expressingcells remained stable. Immunoprecipitation experiments with E^(RNS)- andE2-specific mabs showed a protein pattern identical to that of BVDVinfected PT cells, where gylocprotein E1 is co-precipitated with theE2-specific mab (data not shown). From these data and those obtained bynucleotide sequencing it was concluded that all BVDV structural proteinswere constitutively expressed in PT_(—)805 cells. The cell line was usedfor trans-complementation experiments and for the generation ofinfectious virions as well as for packaging and transducing deletedreplicons.

Recovery and Characterization of Trans-Complemented Recombinant BVDV

For trans-complementation experiments, in vitro-transcribed BVDVreplicon RNA or full-length NCP7 RNA was transfected into monolayers ofPT_(—)805 cells by electroporation. Cell culture supernatants werecollected 24h, 48 h and 72h post transfection (p.t.), clarified bycentrifugation (10,000×g, 5 min) and titrated using highly susceptibleKOP-R cells. KOP-R cells (RIE244, CCVL) are cells of a diploid bovineesophageal cell line, obtained from the collection of cell lines inveterinary medicine at the Federal Research Center of Virus diseases ofAnimals, Insel Riems (CCLV). KOP-R cells were selected due to theirsusceptibility to BVDV infection and their suitability for BVDVpropagation. Cells were grown as described above for the PT cells. As anegative control for replication, replication incompetent controls wereused (deletion mutants NCPΔNS5 and NCP7Δ3′ntraatll).

At the day of collection, replication of BVDV was monitored by IFstaining.

Virus titers of the complemented viruses were determined as infectiousunits (IU). Cell culture supernatants were titrated in triplicate inlog₁₀ steps and 1 ml was inoculated onto KOP-R cells seeded in 24-wellplates. After 12 h of incubation at 37° C., cells were washed, detachedwith a trypsin solution, and counted. An aliquot of the cells wasstained by IF using a BVDV NS3-specific mab and analyzed by flowcytometry. Infectious units (IU) were calculated according to theformula:number of cells in the well×% IF-positive cells×dilution factor=titer inIU/ml

For calculations, only wells with >5% and <30% IF-positive cells wereconsidered.

For all replicons and full-length NCP7, NS3 staining following RNAtransfection led to immunofluorescence patterns similar to that obtainedwith PT cells. Between 80% and 95% of transfected cells were reactivewith NS3-specific antibodies. Infectious recombinant BVDV was detectedin supernatants of NCP7ΔC, NCP7ΔE1 and NCP7-transfected PT 805 cells asshown by infection of KOP-R cells. Virus titers of the resultingcomplemented viruses NCP7ΔC_trans and NCP7ΔE1_trans varied between10^(4,0) to 10^(6.6) IU/ml at 24 h p.t. (Table 2). After transfection offull-length NCP7 RNA into complementing PT_(—)805 cells, virus titers ofup to 10^(7.3) IU/ml could be detected at 24 p.t. (Table 2). Therefore,titers of trans-complemented viruses from supernatants collected at 24 hp.t. were approximately 10000-fold higher than virus titers obtainedafter transfection of full-length NCP7 RNA into non complementing PTcells (Table 2). In addition, no complemented BVDV was detected afterpassaging of PT_(—)805 cell culture supernatants transfected with DI9replicon RNAs (Behrens et al., J. Virol., 72, 2364-2372, 1998) at alltimes p.t. (Table 2).

Using IF of KOP-R cells infected with NCP7ΔC_trans and NCP7ΔE1_trans,expression of E^(RNS), E2 and NS3 could be detected (Table 2).Expression pattern indicated that supernatants of transfected PT_(—)805cells contained infectious virus that can infect and replicate in KOP-Rcells. Titration experiments and subsequent IF staining demonstratedthat no cell-to-cell spread occurred, and that neither NCP7ΔC_trans norNCP7ΔE1_trans were able to form NS3-positive plaques or secondaryinfections in these cells (Table 2). At higher dilutions, only singlecells or small foci of cells were positive by IF-staining for NS3.Control experiments with BVDV NCP7 on KOP-R cells resulted in antigenpositive virus plaques as demonstrated for E^(RNS), NS3 and E2.

The infection of KOP-R cells with trans-complemented viruses could beblocked with BVDV-neutralizing antisera (S-BVD_pos, S-BVDE2) and noinfectivity was detected after inoculation of KOP-R cells (100%neutralization). For neutralization of BVDV, 100 μl of virus suspension(10⁴ TCID₅₀/ml) was mixed in a 24-well plate with 50 μl of undilutedserum and incubated at 37° C. After 2 h, KOP-R cells (2×10⁴) weretransferred to the mixture and incubated for 3 days at 37° C.Subsequently, the monolayer was stained for BVDV-NS3 by indirectimmunofluorescence (IF) (19). (S-BVD_pos) was a pooled anti-BVDV serumcollected from cattle experimentally infected with BVDV strain PT810(3). The homologous neutralization titer of this serum was 1:4,096. ForE2-specific neutralization, serum from sheep, immunized with anE2-expressing modified Vaccinia Virus Ankara (MVA) was used (S-BVD_E2;Beer et al., unpublished data). In a control reaction, incubation with aBVDV antibody-free cattle serum (S-BVD_neg) did not result in virusneutralization (Table 3). TABLE 3 Neutralization of wild-type andcomplemented BVD viruses by BVDV-specific antisera IF analysis 3 dayspost inoculation of the neutralization mix BVDV^(a) S-BVD_pos^(b)S-BVD_E2^(c) S-BVD_neg^(d) NCP7 Ø Ø +^(e) NCP7ΔC_trans Ø Ø +NCP7ΔE1_trans Ø Ø +^(a)Complemented viruses were adjusted to a titer of 10⁴ IU/ml.^(b)Cattle serum prepared against BVDV type 1.^(c)Sheep serum specifically directed against BVDV type 1 E2.^(d)BVDV antibody free cattle serum.^(e)Ø = no IF-positive cells, + = more than 100 IF-positive cells/well.

Example 3 Establishment of C-E_(RNS) Expressing PT Cells (PT 875)

In order to do complementation studies with the BVDV replicons, a bovinecell line was established that constitutively expresses the BVDVstructural proteins Capsid and E^(RNS).

The genomic region encoding the structural proteins C and E^(RNS) ofBVDV strain PT810 (41) was cloned after PCR amplfication of theC-E^(RNS) sequence from the synthetic ORF (Syn-ORF, nucleotides 17 to1000; see “Establishment of C-E^(ms)-E1-E2 expressing cells”) encodingC-E^(RNS)-E1-E2. The PCR product was inserted into the pcDNA3.1expression plasmid (Invitrogen) using restriction sites NotI and XbaI,which had been included into the PCR primers.

The resulting construct pcDNA_C-E_(RNS) (1 μg) was used to transfect PTcells with the SUPERFECT reagent (Qiagen). At 2 days after transfection,cell culture medium was changed to DMEM supplemented with 10% bovineserum and 1 mg of G418 (Gibco, Life Technologies) per ml. G418-resistantcolonies were isolated, and replated several times. After G418 selectionand passaging, resistant cell clones were tested for BVDV E^(RNS)expression using mab WB210. Positive cells were cloned, passaged underG418 selection and tested again for E^(RNS) expression. In the IFanalyses, G418-resistant cell clone PT_(—)875 contained more than 80%E^(RNS) expressing cells. The cell line PT_(—)875 was used fortrans-complementation experiments with BVDV replicons with a deletion inthe capsid encoding region.

Trans-Complementation of NCP7_ΔC with PT_(—)875 Cells

Transfection with NCP7_ΔC led to immunofluorescence patterns similar tothat obtained with PT cells. Between 80% and 95% of transfected cellswere reactive with NS3-specific antibodies. Infectious recombinant BVDVwas detected in supernatants of NCP7ΔC-transfected PT 875 cells as shownby infection of KOP-R cells. Virus titers of the resulting complementedviruses NCP7ΔC_trans varied between 10^(3,0) to 10⁵ IU/ml at 24 h p.t.(data not shown).

Example 4 Immunization of Cattle with Trans-Complemented NCP7ΔC: Dataafter Immunization

Material and Methods

Trans-complemented NCPΔ7C replicon for immunization (NCP7_ΔC_trans) Invitro-transcribed NCP7_ΔC RNA was transfected into PT_(—)805 helpercells. Helper cell supernatants were collected at 24 and 48 h posttransfection, and stored at −70° C. Trans-complemented virions weretitrated using KOP-R cells, and titers in IU were determined. Virionpreparations used for immunization were tested for bacterial and viralcontaminants. In addition, by using passaging on KOP-R cells and RT-PCRanalysis, revertant BVDV as well as wild-type BVDV has been excluded.

Animals, Immunization Protocol, Challenge Infection, Sample Collection,and Clinical Observation

Ten healthy cattle (“Simmental” breeds) seronegative to BVDV and BHV-1and aged between 3 to 5 months were used in this experiment. Fiveanimals were vaccinated twice intramuscularly (i.m.) and intranasally(i.n.) 29 days apart. Each animal of the vaccine group (animals no. 173,537, 554, 758, 977) received 4 ml virus suspension containing 10⁶ IU/mlof NCP7_ΔC_trans (2 ml i.n using an aerosol dispenser adapted to a MUTO™syringe, and 2 ml i.m.). Five calves were used as unvaccinated controls(animals no. 97, 99, 172, 610, 611). All animals were challenged at day62 after the first immunization with the wild-type BVDV strain SE5508(Wolfmeier et al., Arch. Virol., 142, 2049-2057, 1997) using an aerosoldispenser adapted to a MUTO™ syringe. Every animal received 1 ml virussuspension per nostril with a titer of 10⁶ TCID₅₀. Nasal swabs and EDTAblood samples were taken daily during the first 10 days after eachimmunization, and after challenge infection (p. chall.). Additionalsamples were collected at day 14, 21, and 28 p. imm./p. chall.,respectively. Serum samples were collected at days 0, 3, 7, 14, 21, 29p. imm./p. chall. from all animals.

Total white blood cell counts (WBC) were determined by size distributionanalyses with a Cell-Dyn3700 analyser (Abbott). A clinical scoreincluding body temperature was determined for 14 days post eachimmunization, and for 28 days post challenge infection.

Serology

Neutralisation Assay

Sera from all animals were tested in a standard serum neutralisationtest (SNT) versus BVDV strains CP7 and SE5508. The sera were inactivated(30 min, 56 CC) and diluted (log₂; 50 μl) using cell culture medium(MEM, 10% FBS) in triplicates in 96-well plates. One hundred TCID₅₀ BVDVin 50 μl were added and the plates were incubated at 37° C. After 2 hourof incubation, 2×10⁴/well BVDV-negative KOP-R cells in 100 μl cellculture medium were added and the plates were incubated for 5 days at37° C. and 5% CO₂. BVDV antibody-positive and -negative sera were usedas controls, and virus titer was confirmed by back-titration (log₁₀,n=8). BVDV infection was detected in the cells by indirectimmunofluorescence staining. Neutralizing titres were calculated as thegeometric mean values of the reciprocal last dilution (n=3) in which noIF signals were observed.

BVDV Antibody ELISAs

The indirect BVDV ANTIBODY ELISA (IDEXX) and the NS3 blocking BVDVANTIBODY CEDI-TEST (Cedi-Diagnostics) were used to determine BVDV—aswell as BVDV NS3-specific antibodies. Both assays were used according tothe manufacturers instructions.

Virus Isolation

Virus was isolated by using co-cultivation of blood leukocytes withKOP-R cells or inoculation of nasal swabs on monolayers of KOP-R cells.After an incubation period of 4 to 6 days the cells were analysed forBVDV NS3 by using IF staining.

Virus Isolation from Blood Leukocytes

EDTA blood containing 10⁷ leukocytes was centrifuged after haemolysis,washed twice with ice-cold PBS and re-suspended in 1 ml PBS. KOP-R cellcultures were inoculated using 10⁶ washed leukocytes (n=2) in 100 μl PBSand incubated for 4 to 6 days. BVDV was detected using IF staining withNS3-specific mab.

Virus Isolation from Nasal Swabs

Nasal swabs (about 100 mg secretion/swab) were stabilised in 1 ml PBScontaining antibiotics, and 5% BVDV antibody free bovine serum.Following vortexing for 20 sec, an 100 μl aliquot was inoculated onKOP-R cell cultures (n=2). After an incubation period of 4 to 6 days,cells were investigated for BVDV NS3-antigen using IF staining.

E^(RNS) Capture ELISA

For the detection of BVDV antigen after challenge infection, bloodsamples from all animals were tested with a commercially availableE^(RNS) capture ELISA system (Checkit BVDV III, Dr. Bommeli AG,Switzerland) according to the instructions of the manufacturer.

Results

Health Status after Application of NCP7_ΔC_Trans

All animals remained healthy after immunization with NCP7_ΔC_trans, andno abnormal clinical score was observed. Neither leukopenia norincreased body temperatures could be determined (data not shown).

Serology After Immunization

All animals were free of BVDV antibodies at the beginning of theexperiment (BVDV antibody ELISAs and neutralization assays versus BVDVtype I and II), and all control animals remained BVDV antibody negativeuntil BVDV challenge infection. After the first immunization withNCP7_ΔC_trans all animals of the vaccine group scored positive forNS3-specific antibodies at day 29 p. imm. (mean value: 66%), but werenegative in an indirect BVDV ELISA (mean value: 0.13). NS3-specificantibody responses could be detected as early as 14 days p. imm. in serafrom vaccinated animals (FIG. 2 a). After the first immunizationneutralizing antibody titers between 1:3 and 1:12 versus the BVDV strainSE5508 could be detected (FIG. 3).

After the second immunization all immunized animals scored positive inboth the NS3 blocking and the indirect ELISA (FIG. 2). Mean ELISA valuesof about 100% (NS3 blocking ELISA) and about 0.8 (indirect ELISA) couldbe detected between days 7 and 29 after the second immunization (FIGS. 2a and b). In the indirect ELISA, a clear booster effect could beobserved (FIG. 2 b).

At the time of challenge infection (day 62 after the first immunization)all vaccinated animals had neutralizing titers against the BVDV genotype1 challenge strain of 1:256 or more than 1:256 (FIG. 3).

Virus Isolation from Blood Leukocytes and Nasal Swabs after Immunization

No replicating BVDV could be isolated in any of the animals afterinoculation of NCP7_ΔC_trans. All virus detection assays with bloodleukocytes and nasal swabs using KOP-R cells scored negative (Table 4).

Clinical score, virus isolation, and BVDV antigen detection afterchallenge infection After challenge infection, neither clinical signs ofinfection, nor leukopenia, nor fever could be detected in any of theanimals vaccinated with trans-complemented replicons (FIGS. 4 a and b).In contrast, most of the naïve control animals developed clinical signsof a BVDV infection with respiratory symptoms (nasal discharge,coughing) and depression (data not shown). In all control animals amarked leukopenia between days 3 and 8 as well as high body temperaturesof more than 40° C. could be observed post challenge infection (p.chall.) (FIGS. 4 a and b).

BVDV challenge virus was isolated from blood leukocytes and from nasalswabs of all control animals between days 2 and 10 p. chall. (Table 5).However, no BVDV could be isolated from blood leukocytes and nasal swabsof vaccinated cattle (Table 5). In addition, E^(RNS) antigen detectionwith a commercial E^(RNS) capture ELISA was negative for all vaccinatedanimals p. chall., but elevated ELISA values were determined for allanimals of the control group between days 5 and 9 (FIG. 5) and threefrom the five control cattle scored positive with an ELISA value of morethan 35% (data not shown).

Serology after Challenge Infection

After challenge infection, all control animals scored positive in theELISA systems (FIGS. 2 a and b) and the neutralization assay (data notshown). BVDV antibodies could be detected from day 14 p. chall. (FIGS. 2a and b). In the vaccinated group, a marked booster effect could bedetected with the indirect ELISA (mean values from 0.8 at day 0 p.chall. to 1.9 at day 28 p. chall.), but not with the NS3-blocking ELISA(FIGS. 2 a and b).

Discussion

All vaccinated animals remained healthy after inoculation ofNCP7_ΔC_trans. No clinical symptoms could be observed, and neither fevernor leukopenia were detected. Following vaccination, no BVDV could bere-isolated from nasal swabs and blood leukocytes. Taken together,occurrence of a revertant NCP7-like virus can be excluded. We thereforeconclude, that NCP7_ΔC_trans is apathogenic for cattle and acts likedefective in second cycle virions (DISC) or replication incompetent“pseudovirions”.

All control animals remained BVDV antibody negative until challengeinfection. In contrast, all vaccinated animals developed NS3-specificantibodies after the first immunization, and scored positive in anindirect BVDV antibody ELISA after booster vaccination withNCP7_ΔC_trans.

Following challenge infection with a genotype 1 BVDV, all controlanimals had a marked leukopenia and fever. In addition, some of thecontrols developed respiratory symptoms and were depressed for severaldays. In contrast, none of the vaccinated animals developed any clinicalsigns, or leukopenia, or fever after challenge infection. In addition,challenge virus was re-isolated from all control animals for severaldays from blood leukocytes and nasal swabs. However, no BVDV could beisolated from any of the vaccinated animals. We therefore conclude, thatimmunization with NCP7_ΔC_trans provides complete protection from BVDVclinical signs, viremia and virus shedding. TABLE 4 Virus isolation fromblood leukocytes and nasal swabs following immunization withNCP7_ΔC_trans days post second immunization days post first immunization0 1 2 3 4 5 6 7 8 9 10 Animal No. 0 1 2 3 4 5 6 7 8 9 10 29 30 31 32 3334 35 36 37 38 39 173 0^(a)/0^(b) 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 537 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 5540/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 758 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 977 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/00/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0^(a)number of positive inoculations with 1 × 10⁶ leukocytes (2inoculations per sample; no positive samples = white, 1 positive sample= light grey, 2 positive samples = dark grey)^(b)number of positive inoculations with 10 mg nasal secretion (2inoculations per sample; no positive samples = white, 1 positive sample= light grey, 2 positive samples = dark grey)

TABLE 5 Virus isolation from blood leukocytes and nasal swabs followingchallenge infection with BVDV SE5508

^(a)number of positive inoculations with 1 × 10⁶ leukocytes (2inoculations per sample; no positive samples = white, 1 positive sample= light grey, 2 positive samples = dark grey)^(b)number of positive inoculations with 10 mg nasal secretion (2inoculations per sample; no positive samples = white, 1 positive sample= light grey, 2 positive samples = dark grey)

1. A replicon of a pestivirus which is incapable of expressing one ormore structural proteins of the virus, characterized in that saidreplicon expresses all structural proteins of a pestivirus except for afunctional C and/or E1 protein, but wherein the coding sequencesencoding the part of the C and/or the E1 protein essential for furtherdownstream processing are retained or replaced by a coding sequenceencoding analogous signal sequences from another pestiviral species. 2.The replicon according to claim 1, characterized in that at least partof the coding sequence of the E1 or C protein has been deleted from saidreplicon.
 3. The replicon according to claim 1, characterized in thatsaid replicon does not encode a functional C protein.
 4. The repliconaccording to claim 1, characterized in that said replicon is of theBovine Viral Diarrhea Virus (BVDV).
 5. The replicon according to claim4, characterized in that the coding region encoding amino acid positions201-243 of the C protein have been deleted.
 6. The replicon according toclaim 1, characterized in that said replicon does not encode afunctional E1 protein.
 7. A The replicon according to claim 4,characterized in that the coding region encoding amino acid positions498 to 653 of the E1 protein have been deleted.
 8. The infectious viralparticle of Pestivirus, characterized in that it contains a repliconaccording to claim
 1. 9. A method for the production of viral particlesof a Pestivirus according to claim 8, characterized in that said methodcomprises the following steps: a. Providing cells that are permissivefor the Pestivirus and express Pestiviral E1 and/or C protein, b.Transfecting said cells with in-vitro transcribed RNA of a repliconaccording to claim 1, c. Culturing transfected cells obtained in step b,d. Harvesting the viral particles from the cultured cells.
 10. Themethod according to claim 9, characterized in that said pestivirus isBVDV.
 11. The method according to claim 10, characterized in that saidcells express the E1 and/or C protein of BVDV.
 12. A vaccine containinginfectious viral particles according to claim 8 and a pharmaceuticallyacceptable carrier.